Delivery of compounds with rehydrated blood cells

ABSTRACT

Fixed-dried blood cells carrying an active agent are described, along with methods of making the same, methods of using the same, and compositions containing the same. The blood cells are preferably blood platelets.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/845,045, filed May 13, 2004, and claims the benefit of U.S.Provisional Patent Application Ser. No. 60/533,059, filed Dec. 29, 2003,and U.S. Provisional Patent Application Ser. No. 60/471,005, filed May16, 2003, the disclosures of which are incorporated by reference hereinin their entirety.

This application is also related to U.S. patent application Ser. No.11/751,295, filed May 21, 2007; and U.S. patent application Ser. No.11/149,515, filed Jun. 10, 2005; which are divisional and continuationapplications, respectively, of application Ser. No. 10/845,045; thedisclosures of which are incorporated by reference herein in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support from the Department ofDefense and the National Institutes of Health under Grant Numbers1-P20-DE123474, 1-P60-DE 130789, and 1 R21 EB002863. The United StatesGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns methods and compositions for deliveringactive agents to a subject in need thereof and more particularly tofixed-dried blood cells that carry an active agent and that are usefulto deliver active agents to a site of interest.

BACKGROUND OF THE INVENTION

Platelets have been recognized for decades as potential tools forcarrying therapeutics to sites of vascular injury. However, thepractical utility of platelets as therapeutic delivery vehicles has beenlimited because platelets must be freshly isolated, modified with thetherapeutic, and then infused in a short time-span. The utility ofcryopreserved platelets and normally liquid-stored for therapeuticdelivery is limited by storage lesion and, in the case of frozenplatelets, the need to remove cryopreservatives. Hence the practicalapplication of cryopreserved platelets, as well as preserved blood cellsin general, has been limited.

SUMMARY OF THE INVENTION

A first aspect of the present invention is fixed-dried blood platelets,red blood cells (RBCs,) or combinations thereof carrying a heterologouscompound, active agent or compound of interest, along with compositionscomprising, consisting of or consisting essentially of the same. Suchplatelets, RBCs or combinations thereof are preferably mammalian bloodcells such as human blood platelets and RBCs. The compound or activeagent may be associated with the platelets and/or RBCs in any manner,such as coupling or by being contained within the platelets and/or RBCs.The platelets and/or RBCs may be aldehyde-fixed, and in one embodimentthe platelets are characterized in that they adhere to thrombogenicsurfaces; undergo shape change upon adhering to a thrombogenic surface;lead to the formation of a hemostatic plug upon adhering to athrombogenic surface; and release their granular contents. The activeagent or compound to be delivered may be an antiviral agent such as anucleoside analog antiviral agent (for example ribavirin), a bloodcoagulation protein such as Factor VII, a nucleic acid (e.g., DNA, RNA),a detectable compound such as a detectable protein or peptide, etc.

A second aspect of the present invention is a pharmaceutical compositioncomprising, consisting of or consisting essentially of for example, from0.01 or 0.1 to 99.9 or 99.99 percent by weight of a pharmaceuticallyacceptable carrier (e.g., an aqueous or nonaqueous carrier; a solid,liquid or gel carrier, etc.); and, for example, from 0.01 or 0.1 to 99.9or 99.99 percent by weight of fixed-dried blood cells carrying an activeagent as described herein, the fixed-dried blood cells optionallyrehydrated in the pharmaceutically acceptable carrier.

A further aspect of the present invention is a method of makingfixed-dried blood cells for delivering a compound of interest or anactive agent to a subject in need thereof, comprising: providing fixedblood cells (typically mammalian cells and preferably human cells,particularly RBCS, platelets, and combinations thereof) carrying theactive agent; associating the active agent to the fixed blood cells; anddrying the fixed blood cells (e.g., by freeze-drying or lyophilization)to produced fixed-dried blood cells carrying the active compound. Themethod may further comprise the step of rehydrating the fixed-driedblood cells in a pharmaceutically acceptable carrier to provide apharmaceutically acceptable composition comprising rehydratedfixed-dried blood cells carrying the active agent. The associating stepmay be carried out by any suitable means, such as by coupling the activeagent to the cells or introducing the active agent into the cells. Thecells may be aldehyde-fixed platelets, and in one embodiment the cellsare platelets that are characterized in that, upon reconstitution, they:adhere to thrombogenic surfaces; undergo shape change upon adhering to athrombogenic surface; lead to the formation of a hemostatic plug uponadhering to a thrombogenic surface; and release their granular contents.

A further aspect of the present invention is a method of delivering acompound of interest to a thrombogenic surface (e.g., in a subject, orto a tissue in vitro for diagnostic or compound testing purposes),comprising: providing fixed-dried blood cells carrying the compound ofinterest as described herein; rehydrating the fixed-dried blood cells inan acceptable carrier (e.g., a pharmaceutically or physiologicallyacceptable carrier) to provide a composition comprising rehydratedfixed-dried blood cells carrying the active agent; and thenadministering the pharmaceutical composition (e.g., to the subject, tothe tissue in vitro) so that an effective amount of the compound ofinterest is delivered to a thrombogenic surface. The subject istypically a mammalian subject such as a human subject, and the compoundof interest may be a diagnostic or therapeutic agent.

A further aspect of the present invention is a method of delivering acompound of interest to macrophages of the RES in a subject. The subjectis typically a mammalian subject such as a human subject, and thecompound of interest may be a diagnostic or therapeutic agent.

A further aspect of the present invention is a method of delivering anactive agent to a site of interest (e.g., to a subject or to a tissue invitro), comprising: providing fixed-dried blood cells carrying theactive agent as described herein; rehydrating the fixed-dried bloodcells in a pharmaceutically acceptable carrier to provide apharmaceutically acceptable composition comprising rehydratedfixed-dried blood cells carrying the active agent; and thenadministering the pharmaceutical composition to the site of interest(e.g., to a subject or to a tissue in vitro), so that an effectiveamount of the active agent is delivered to said site of interest. Theeffective amount of active agent may be less than 15% (15 percent) ofendogenous platelet equivalent. The subject is typically a mammaliansubject such as a human subject, and the active agent may be adiagnostic or therapeutic agent. The cells may be aldehyde-fixed cells,and the subject may for example be afflicted with a vascular injury.

The foregoing and other objects and aspects of the present invention areexplained in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the proton NMR Spectra of nucleoside copolymers.

FIG. 2 shows the fluorescent microscopy of polymer-modified RLplatelets.

FIG. 3 shows ristocetin aggregation of control and polymer modified RLplatelets.

FIG. 4 schematically illustrates the preparation of Platelet/AAV˜Lac Zreporter conjugates.

FIG. 5 shows flow cytometric analysis of rFVIIa-FITC bound to RLplatelets.

FIG. 6 shows that rFVIIa binding to RL:platelets is concentrationdependent.

FIG. 7 shows that rFVIIa is active on the surface of RL platelets.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Subjects to be treated with the methods and compositions of the presentinvention include both human subjects and animal subjects for veterinaryand drug development purposes. Animal subjects are, in general,mammalian subjects, including but not limited to pig, sheep, cow, horse,goat, cat, dog, mouse and rat subjects.

“Thrombogenic surface” as used herein refers to any natural orartificial thrombogenic surface, including but not limited to woundtissue, blood vessel plaques such as atherosclerotic plaques, activatedendothelium due to local or systemic inflammation, vessels, surfaces ortissues that are rendered thrombogenic in a subject as a toxicside-effect due to administration of anticancer, antineoplastic orantiproliferative agent, surfaces of foreign or implanted items in thesubject, including metals, polymers, etc., as found in stents,catheters, biomedical implants such as pacemakers and leads, orthopedicimplants such as artificial joints, etc. A compound may be administeredto a thrombogenic surface for any purpose, such as for therapeuticpurposes or diagnostic purposes (e.g., imaging or detection of athrombogenic surface by any suitable means such as radioimaging, tissuebiopsy, implant removal and determination of whether the deliveredcompound is found on the implant surface, etc.).

“Platelets” utilized in carrying out the present invention are, ingeneral, of animal, and preferably mammalian, origin (e.g., pig, sheep,cow, horse, goat, cat, dog, mouse, rat, human, etc.). Platelets may bederived from the same species into which the platelets are introduced,or from a different species from which the platelets are introduced. Inone embodiment, platelets are harvested from a subject, used to preparethe active agents described herein, and after being so prepared areadministered at a later time back to the same subject from which theplatelets were harvested.

“Red blood cell” as used herein includes any erythrocyte of mammalianorigin (e.g., pig, sheep, cow, horse, goat, cat, dog, mouse, rat, human,etc.). Red blood cells may be derived from the same species into whichthe red blood cells are introduced, or from a different species fromwhich the red blood cells are introduced. In one embodiment, red bloodcells are harvested from a subject, used to prepare the active agentsdescribed herein, and after being so prepared are administered at alater time back to the same subject from which the red blood cells wereharvested.

“Fixed blood cells” herein refers to blood cells, which may beplatelets, red blood cells, or mixtures thereof, which have beenchemically treated with at least one chemical compound that isincorporated into at least a portion of the cells to structurallystabilize and/or extend the shelf-life of the cells.

“Fixed-dried blood cells” herein refers to blood cells, which may beplatelets, red blood cells, or mixtures thereof, which have been fixed,and additionally have had water removed therefrom by any suitabletechnique such as drying, dehydrating, lyophilizing or freeze-drying,etc., to further structurally stabilize and/or extend the shelf-lifethereof.

“Rehydrated fixed-dried blood cells” refers to a fixed-dried bloodcells, which may be platelets, red blood cells, or mixtures thereof,which has been contacted to or combined with an aqueous solution so thatwater is taken up into the intracellular space.

“Blood coagulation protein” as used herein includes any suitable bloodcoagulation protein, including but not limited to Factor VII, Factor IX,Factor X, as well as coagulation proteins that generate Factor VII orFVIIa, such as Factor XII or Factor XIIa, or Factor X or Factor Xa,Protein C, Protein S, and Prothrombin. Such proteins may be natural orsynthetic and include proteins containing minor modifications of thenaturally occurring protein (i.e., analogs). Where naturally occurringthe protein may be of any species of origin, preferably mammalian orhuman as described herein, and in one embodiment is of the same speciesof origin as the subject to which it is administered.

“Blood anti-coagulation protein” as used herein includes any suitableblood anti-coagulation protein, including but not limited to activatedprotein C, protein S, heparin and heparinoids, etc. Such proteins may benatural or synthetic and include proteins containing minor modificationsof the naturally occurring protein (i.e., analogs). Where naturallyoccurring the protein may be of any species of origin, preferablymammalian or human as described herein, and in one embodiment is of thesame species of origin as the subject to which it is administered.

“AAV” as used herein refers to “adeno-associated virus”.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, etc.

The term “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

Applicants specifically intend that the disclosures of all United Statespatent references cited herein be incorporated by reference herein intheir entireties.

1. Platelets.

Platelets may be fixed in accordance with known techniques, such asdescribed in U.S. Pat. Nos. 4,287,087; 5,651,966; 5,902,608; 5,891,393;and 5,993,084. In general, such methods involve providing bloodplatelets (e.g., human, mammalian); contacting said human platelets to afixative (e.g., an aldehyde) for a time sufficient to fix saidplatelets; removing said fixative from said platelets; and then dryingsaid platelets to produce fixed-dried blood platelets. In a preferredembodiment, the contacting step is carried out for a time sufficient tokill said microorganisms. In a preferred embodiment, the contacting stepis carried out for a time insufficient to cause said platelets to losethe capability, upon reconstitution, to; (i) adhere to thrombogenicsurfaces; (ii) not adhere to non-thrombogenic surfaces; (iii) undergoshape change (spreading) upon adhering to a thrombogenic surface; (iv)adhere to one another to form a hemostatic plug upon adhering to athrombogenic surface; and (v) release their granular contents.

More particularly, fixed-dried blood platelets for use in the presentinvention may be fixed with a compound selected from the groupconsisting of formaldehyde, paraformaldehyde and glutaraldehyde, withparaformaldehyde currently preferred. In general, washed platelets maybe fixed by incubating them, typically at room temperature, for up to 60minutes in a solution of up to 1.8% aldehyde. An alternative techniqueis to fix platelets by incubating the platelets in a permanganatesolution (e.g., sodium permanganate, potassium permanganate). Ingeneral, washed platelets may be prepared by this technique byincubating them for from 5 to 20 minutes in from 0.001 to 1 g/dL ofKMnO₄ or NaMnO₄ solution, more preferably by incubating them for from 5to 15 minutes in from 0.005 to 0.5 g/dL of KMnO₄ or NaMnO₄ solution,

Blood platelet preparations for use in preparing pharmaceuticalformulations should be essentially free of extraneous matter,particularly lysed blood platelets which would present free thrombogenicagents to a patient administered the preparation. Hence, care must betaken to sufficiently fix the platelets (preferably without destroyingthe viability thereof, as indicated by the characteristics set forthabove) prior to drying, as undue lysis will otherwise occur during thedrying step. For example, platelet preparations suitable for use inpreparing human pharmaceutical formulations preferably show, onreconstitution of 10⁹ platelets in one milliliter of solution, less than10×10⁶ microparticles (the fragmentary remains of lysed platelets) permilliliter, and preferably show less than 150 International Units (IU)per liter of lactate dehydrogenase in the supernatant after resuspensionand pelleting (where 2200 IU per liter represents total lysis of 10⁹cells in 1 milliliter).

Drying of platelets after fixation may be carried out by any suitablemeans, but is preferably carried out by lyophilization. Care should betaken to stabilize the platelet preparation prior to drying as anunacceptable level of platelet lysis may otherwise occur. Stabilizationmay be carried out by suspending the platelets in a solution containinga suitable water replacing molecule (or “stabilizer”), such as albuminor trehalose, and then drying the solution. In one embodiment, from 0.1to 20 percent by weight albumin is employed, more preferably from 1 to10 percent by weight albumin is employed, and most preferably from 5 to10 percent by weight albumin is employed. For administration to asubject, the albumin in the preparation should be of the same species asthe subject (e.g., human albumin). In the alternative, the preparationmay be dried with albumin of a different species, the albumin separatedfrom the platelets on reconstitution, and albumin of the same speciesadded back to the reconstituted preparation for administration to thesubject, but care should be taken to remove all non-species specificalbumin as it may be antigenic in the subject being treated. Once dried,the platelets may be coupled or associated with a compound to bedelivered to produce an “active agent” of the present invention, asdescribed further below.

2. Lyophilized RBCs.

Cross-linked and lyophilized RBCs of the present invention may beprepared with bifunctional cross-linking reagents that are homo orheteromeric with reactive the following reactive moieties: aldehydes,ketones, hydrazides, N-hydroxysulfosuccinimides, N-hydroxysuccinimides,maleimides, imidoesters, active halogens, pyridyl-disulfides,isocyanates, nitrobenzoyloxysuccinimides, nitrobenzenes, imidoesters,photo-activatable azidophenyls and azidopolyaromatics, as well aszero-spacer carbodimide catalysts. Multi (poly) functional reagents arealso considered, as are combinations of two or more cross-linkers,either serially reacted with RBCs for reacted together with RBCs.

RBCs are obtained from mammalian blood with standard phlebotomy,apheresis or exsanguination methods according to approved IACCOCprotocols. RBCs are freed from plasma platelets, leukocytes and plasmaproteins my differential centrifugation, and then treated with chemicalcross-linkers. The reaction of the RBCs with the cross-linkers are ingeneral carried out for defined periods of time at temperatures between20° C. and 37° C. at pre-determined concentration of RBCs. As discussedin greater detail below, care must be taken to sufficiently fix theplatelets or undue lysis will be measured upon rehydration of thelyophilized product. The cross-linking step can be carried out in thepresence of antioxidants and free-radical scavengers, and thecross-linking reaction can be quenched by adding compounds that containprimary amines. After cross-linking, the RBCs are removed from excesscross-linker and reaction products with differential centrifugation,chromatography and/or dialysis.

Freezing of RBCs after cross-linking may be carried out over a widerange of cooling rates at ambient or hyperbaric pressures. If RBCs (withzero or reduced concentrations of cross-linkers) are frozen into thehigh-pressure phase states of ice (e.g., ice II/III) samples arepreferably isothermally pressurized and then isobarically cooled tounder −120° C., the point at which ice II/III is metastable. RBCs can befrozen in the presence of “stabilizer” small molecules (e.g., glycerol),proteins (e.g., albumin) and polymers (e.g., PEG) which substitute forwater in the ice crystal matrix. The preferred “stabilizer” is PEG 8,000at a final concentration of 1% (w.v). The type and level of “stabilizer”must be infusible as rehydrated. Lyophilization is carried out fromtemperatures below 0° C., preferably 40° C. if the RBCs were frozen atambient pressure for ice I, and near or less than −120° C. for moleculardistillation from the ice II/III phase states.

The chemical modification of RBC membranes with cross-linkers imparts a“foreign” nature to the cells with respect to recognition by thereticuloendothelial system and thrombogenic with respect to contactactivation of platelets. The surface membrane is thus occluded bycovalently attaching polymers that sterically coat the cell membrane.Polymers, particularly water-soluble polymers, that may be used to carryout the present invention are, in general, naturally occurring polymerssuch as polysaccharides, or synthetic polymers such as polyalkyleneoxides such as polyethylene glycols (PEG), polyalkylene glycols,polyoxyethylated polyols, polyvinylpyrrolidone, polyacrylates such aspolyhydroxyethyl methacrylate, polyvinyl alcohols, and polyurethane. Thepolymers may be linear, branched or dendrimeric and may be substitutedor unsubstituted. The polymers may, as noted above, be hydrophilic,lipophilic, or both hydrophilic and lipophilic. Polymers are covalentlyattached through the membrane through reactive chemical functions thatinclude, but are not limited to, aldehydes, ketones, hydrazides,N-hydroxysulfosuccinimides, N-hydroxysuccinimides, maleimides,imidoesters, active halogens, pyridyl-disulfides, isocyanates,nitrobenzoyloxysuccinimides, nitrobenzenes, imidoesters,photo-activatable azidophenyls and azidopolyaromatics, as well aszero-spacer carbodimide catalysts. The preferred polymer is PEG 5,000with a terminal aldehyde for covalent attachment to surface lysines viaSchift's base formation. We have found that most of the washing andfixation steps for a larger-scale red cell preparation canadvantageously be performed in a closed system by utilizing an appliancecalled the IBM 2991 Cell Washer, which was originally designed for bloodbanks to facilitate washing of frozen red cell units to removecryoprotectant agents like DMSO or glycerol just prior to transfusion.The advantage to employing this device for our purposes in preparingfreeze-dried red blood cells is that it provides an aseptic environmentfor the multiple steps of washing and fixation which would otherwiserequire handling of the red cells in an open container, and the CellWasher induces less shear stress to resuspend the packed red cells aftereach step than would be experienced with a resuspension method by hand.As an example, we introduce into the Cell Washer processing bag a volumeof 150-200 mL of a suspension of leukodepleted red blood cells at a cellcount of 3-4×10⁹/mL and attach the tubing harness as described in theoperating instructions. Then we introduce a volume of 200-250 mLphosphate washing buffer containing 0.1% BSA sterilely through thetubing harness and perform wash cycle #1. At the end of the agitationand spinning period programmed into the 2991, the supernatant washingfluid is automatically expressed out to waste and fresh buffer isintroduced thru the harness for a total of three washes. After thespinning step of the third wash, a fixation solution containing 0.05%glutaraldehyde in Hank's buffered salt solution [HBSS] is introduced fora timed incubation of 20 minutes at room temperature before spinning,and then three more washing steps are performed with the phosphatebuffer. At this point the fixed, washed red cell suspension is removedfrom the 2991 processing bag and handled in vials and bottles for thebulking and freeze-drying steps.

3. Compounds to be Delivered.

Examples of compounds that may be coupled to platelets to produce activeagents of the present invention include, but are not limited to, nucleicacids such as RNA, DNA, proteins or peptides (enzymes, antibodies,etc,), viruses, bacteria, small organic compounds (e.g., monomers),synthetic and semisynthetic polymers, nanoparticles, chelated metals andions, etc. Such compounds may have any suitable function or activitydepending upon the particular object of the treatment or method,including but not limited to antimicrobial, antibacterial, or antiviralactivity; blood coagulation or anti-coagulation activity; reporter ordetectable activity, etc. Additional examples of compounds that may becoupled to platelets to produce active agents of the present inventioninclude, but are not limited to, vasoactive, antioxidant,antithrombotic, anticarcinogenic, antiatherogenic, antimitotic,antiangiogenic, and antiproliferative compounds. It will be appreciatedthat all such compounds can be contained within a vesicle, micelle orother particle which is in turn associated with or coupled to theplatelet.

Antiviral Compounds.

In one embodiment of the present invention, the compound carried by theplatelets is an antiviral compound. Any suitable antiviral compound canbe used, including but not limited to sialic acid analogues, amantadine,rimantadine, zidovudine, vidarabine, idoxuridine, trifluridine,foscarnet, etc. In one embodiment the antiviral compound is a purinenucleoside analog, examples of which include but are not limited toacyclovir, didanosine, ribavirin, ganciclovir, and vidarabine, andantisense nucleosides, RNA and DNA.

In one embodiment of the invention, platelets carrying antiviralcompounds are administered to patients afflicted with a viral infectionin an amount sufficient to treat the viral infection. In one embodimentthe patient is infected with a hemorrhagic fever viruses, such as avirus of the Filoviridae, Arenaviridae, Bunyaviridae or Flaviviridaefamilies.

Blood Coagulation and Anticoagulation Proteins.

The compound to be delivered may be a drug coagulation protein, such asFactor VII, Factor IX, Factor X, Factor XII, Protein C, Protein S,Prothrombin, anticoagulation proteins such as activated protein C,protein S, heparin and heparinoids, and pro- or anti-coagulationproteins of reptile or insect origin, and others.

Such compounds are known. For example, Factor VII or Factor VIIa whichmay be utilized in carrying out the present invention (this termincluding modified Factor VII or Factor VIIa or Factor VII analogs whichretain the blood coagulation activity of Factor VII) is described in,among others, U.S. Pat. Nos. 6,461,610; 6,132,7306,329,176; 6,183,743;6,186,789; 5,997,864; 5,861,374; 5,824,639; 5,817,788; 5,788,965;5,700,914; 5,344,918; and 5,190,919. In one embodiment, recombinanthuman Factor VIIa is preferred.

In one embodiment of the invention, platelets carrying blood coagulationproteins are administered to a subject afflicted with a wound in anamount effective to promote blood coagulation at the wound and/orhealing of the wound.

Nucleic Acids.

Nucleic acids to be carried by platelets of the present invention mayencode a detectable protein or peptide such as Lac-Z,beta-glucuronidase, horseradish peroxidase, a fluorescent protein suchas green fluorescent protein, etc.

In one embodiment of the invention, platelets carrying nucleic acidsencoding a detectable protein are administered to a subject in an amounteffective to express the detectable protein in atherosclerotic tissue orplaques in blood vessels to thereby produce an improved animal model ofatherosclerosis. Such animals (which preferably are animals that, bydiet and/or breeding are susceptible to atherosclerosis) may beadministered a putative antiatherogenic compound, and/or antiatherogenicdiet, and then compared to a control animal that has not beenadministered the putative anti-atherogenic compound and/oranti-atherogenic diet, and the extent of atherogenic plaque formation inexperimental animals versus control animals compared by visualizingplaques through expression of the detectable protein.

In other embodiments of the invention, the nucleic acid may encode atherapeutic protein or peptide. Examples include, but are not limitedto, nucleic acids encoding an anti-atherogenic protein or peptide suchas DNA encoding the ras binding domain (RBD) of the ras effector proteinRGL₂ (e.g., to inhibit proliferation), DNA encoding endothelial nitricoxide sythetase variants (e.g., to inhibit platelet function), DNAencoding the NF-κB super-repressor (e.g., to inhibit inflammatoryprocesses).

4. Associating Compounds with Platelets.

Compounds to be delivered may be associated with platelets by anysuitable technique, including but not limited to: (1) directlychemically coupling the compound to be delivered to the platelet surfacemembrane; (2) conjugating the compound to be delivered to a polymer thatis in turn coupled to the platelet's internal membrane; (3)incorporating the compound to be delivered into unilamellar ormultilamellar phospholipid vesicles that are in turn internalized intothe platelets; (4) absorbing or internalizing the compound to bedelivered into nanoparticles, e.g., buckminsterfullerene, that are inturn internalized into the platelets; (5) coupling the compound to bedelivered to proteins that are internalized for trafficking to alphagranules in the platelets; (6) coupling the compound to be delivered toproteins (or other macromolecules) or particles that are phagocitized bythe platelets; (7) hydrophobically partitioning the compound to bedelivered into membranes; (8) physically entrapping the compound to bedelivered in the platelet intracellular space through pores that areformed with electrophoration, complement treatment, lytic proteinexposure, etc; (9) adsorbing the compound to the exterior surface of thecell by non-covalent physical or chemical adsorption, that are in turninternalized into the platelets.

Cross-Linking Chemistry for Preparing Compound-Platelet Conjugates.

Compound-platelet conjugates in which platelets are coupled to proteins,antiviral compounds or the like may be prepared with homo- orhetero-bifunctional cross-linking reagents that can contain, but are notlimited to, the following reactive moieties: aldehydes, ketones,hydrazides, N-hydroxysulfosuccinimides, N-hydroxysuccinimides,maleimides, imidoesters, active halogens, pyridyl-disulfides,isocyanates, nitrobenzoyloxysuccinimides, nitrobenzenes, imidoesters,photo-activatable azidophenyls and azidopolyaromatics, as well aszero-spacer carbodimide catalysts. Multi (poly) functional reagents withon or more of these moieties are also considered, as are combinations oftwo or more cross-linkers, either serially reacted or reacted inconcert. The cross-linking step can be carried out in the presence ofantioxidants and free-radical scavengers, and the cross-linking reactioncan be quenched by adding compounds that contain primary amines. Aftercross-linking, excess reagent can be removed with methods that includebut are not limited to with differential centrifugation, chromatographyand/or dialysis.

Viral Encapsidation.

In the case of nucleic acids to be delivered, the nucleic acid may beencapsidated or enclosed within a viral capsid or particle, which viralcapsid or particle may in turn be conjugated or coupled to theplatelets. One suitable virus into which the nucleic acid may beencapsidated is the adeno-associated virus (AAV). The AAV virus is knownand a nucleic acid of interest to be delivered may be encapsidated orpackaged therein in accordance with known techniques. See, e.g., U.S.Pat. Nos. 6,548,286; 6,491,907; 6,489,162; 6,458,587; 6,410,300;6,268,213; 6,204,059; 6,093,570; 6,057,152; 6,040,183; 5,869,305;5,863,541; 5,773,289; 5,753,500; 5,478,745; 5,436,146; and 5,139,941. Inaddition to AAV, it will be appreciated that other viruses, includingbut not limited to such as adenoviruses, lentiviruses, hepatitisviruses, herpesviruses, can also be used to encapsidate a nucleic acidfor association with a platelet to carry out the present invention.

Once the nucleic acid of interest is encapsidated or packaged in a viralparticle, viral particles may be associated with or coupled to plateletsin accordance with known techniques.

In general, the compound to be delivered is coupled to or associatedwith the platelets so that each platelet carries, or has associatedtherewith, at least 1,000, and more preferably at least 10,000,individual molecules of the compound to be delivered.

Once prepared, the platelets with associated compound to be deliveredcomprise an “active agent” which may be stored in frozen form,refrigerated, or at room temperature (depending upon the shelf liferequired) for subsequent reconstitution and use.

5. Reconstitution and Administration of Active Agents.

Pharmaceutical formulations of the present invention may simply comprisedried (preferably lyophilized) blood cells carrying the active agent,pyrogen-free and sterile in a sterile aseptic package. Albumin may beincluded, as noted above. Pharmaceutical formulations may also comprisea platelet preparation of the present invention reconstituted in apharmaceutically acceptable carrier. Additional agents, such as buffers,preservatives, and other therapeutically active agents, may also beincluded in the reconstituted formulation. See, e.g., U.S. Pat. No.4,994,367 (the disclosure of which is incorporated herein by reference).The amount of blood cells and pharmaceutical carrier is not critical andwill depend upon the particular application of the blood cells, whetheror not they have been rehydrated, etc., but in general will range from 1or 10 percent by weight blood cells up to 90 or 99 or even 99.9 percentby weight blood cells, and from 0.01, 1 or 10 percent by weight ofpharmaceutically acceptable carrier up to 90 or even 99 percent by weighpharmaceutically acceptable carrier. In some embodiments of fixed-driedblood cells that have not been rehydrated the composition may consistessentially of or consist entirely of the fixed-dried blood cells, freeof any particular carrier.

The fixed-dried blood cells described above may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the fixed-dried blood cells may be admixedwith, inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid (including powders), and is preferably formulatedwith the blood cells and packaged as a unit-dose formulation, forexample, a bottle of lyophilized powder which may be reconstituted byaddition of an aqueous solution.

For rehydrating the blood cells, any aqueous carrier which rehydratesthe platelets so that they possess the characteristics enumerated aboveand are suitable for intravenous injection may be used (e.g., sterile,pyrogen free, physiological saline solution). For example, an aqueouscarrier may be injected into a bottle containing a pharmaceuticalformulation of the invention in the form of a lyophilized powder, thecontents agitated if necessary, and then the pharmaceutical formulationin the form of an aqueous suspension of rehydrated fixed-dried bloodcells withdrawn from the bottle and administered by injection into apatient.

The compounds of the invention are preferably administered internally,e.g., orally or intravenously, in the form of conventionalpharmaceutical compositions, for example in conventional enteral orparenteral pharmaceutically acceptable excipients containing organicand/or inorganic inert carriers, such as water, gelatin, lactose,starch, magnesium stearate, talc, plant oils, gums, alcohol, Vaseline,or the like. The pharmaceutical compositions can be in conventionalsolid forms, for example, tablets, dragees, suppositories, capsules, orthe like, or conventional liquid forms, such as suspensions, emulsions,or the like. If desired, they can be sterilized and/or containconventional pharmaceutical adjuvants, such as preservatives,stabilizing agents, wetting agents, emulsifying agents, buffers, orsalts used for the adjustment of osmotic pressure. The pharmaceuticalcompositions may also contain other therapeutically active materials.The pharmaceutical compositions of the invention can be made usingconventional methods know in the art of pharmaceutical manufacturing.

Reconstituted pharmaceutical formulations of the present invention aretypically administered to human patients by parenteral administration(e.g., intravenous injection, intraarterial injection). The amount ofthe pharmaceutical formulation administered will vary depending upon theweight and condition of the patient, but will typically range from 20 to350 milliliters in volume, and from 1×10⁹ to 3×10⁹ platelets permilliliter (and more preferably from 2×10⁹ to 3×10⁹ platelets permilliliter) in concentration. Pharmaceutical formulations may bepackaged in a sterile, pyrogen free container to provide these volumesand dosages as a unit dose. The particular route of administration isnot critical and will depend upon the condition being treated, withtopical administration or application into penetrating wound sites alsobeing utilized for such injuries and their corresponding treatments.

Three applications, involving wound site imaging, coagulation factordelivery and antiviral delivery, are among those considered in thefollowing discussion.

a) RL platelets for wound localization with magnetic resonance imaging—

RL platelets can be loaded with paramagnetic nanoparticles that canfunction as MRI contrast agents when the rehydrated cells localize tothe sites of vascular injury. Two recent advances have provided thetools to prepare RL platelet-MRI contrast agent formulations. First,infusible gadolinium (Gd)-chelates have been approved by the FDA (e.g.,Magnevist™) and are proving useful, as a relaxation contrast agent, forthe evaluation of vascular pathologies (e.g., Rofsky and Adelman, 2000).A wide range chemically activated of Gd-chelators for attachment toprimary amines on macromolecules are available, and have lead to thedevelopment of research probes for specific angiographic imagingapplications (see Artemov, 2003, for a review). For example, fibrinclots have been localized with Gd-anti-fibrin antibody probes for thedetection of vulnerable plaques (Flacke et al., 2001).

Secondly, major advances have been made in the preparation of magneticnanoparticles (see Pankhurst et al, 2003, for a review). Of particularimportance is the synthesis of iron oxide (maghemite and magnetite)nanoparticles with narrow size distribution (Chatterjee et al, 2003).Furthermore, methods for the modification of iron oxide particles withpolymers for the attachment of macromolecules have been developed(Chatterjee et al. 2003) and used to isolate blood cells in magneticfields (Chen, et al., 2000). The RL platelet represents a uniqueplatform for targeting iron oxide nanoprobe fabrication and Gd-chelatesto sites of vascular injury.

b) RL platelet-coagulation factor conjugates for hemostasis incoagulopathic conditions—

The tethering of coagulation factors (such as FVIIa) to the surface ofRL platelets and/or loading of α-granules etc) with coagulation factors(FXIIIa for clot stabilization, caged thrombin for photoactivation(Arroyo et al., 1997)), is a logical strategy for delivering replacementfactors to vascular wound sites in hemophiliacs that have circulatingantibody inhibitors to standard replacement therapeutics (refractoryhemophiliacs) as well as patients with other types of coagulopathies.Recombinant FVIIa has been hypothesized to provide hemostasis throughtwo mechanisms that are operant on the platelet surface. First, theability of recombinant factor VIIa (rFVIIa) to provide hemostasis inhemophilia patients (Hedner and Glazer, 1992) has been theorized toinvolve the direct activation of factor Xa on the surface of platelets(e.g., see Monroe et al., 1997 and 2000), thus “bypassing” the need forfactors 1× and VIII. This “bypass” activity of the rec FVIIa might bethe functional basis for the utility of the Novo Nordisk product as ahemostatic agent in hemophilia A and B. Secondly, in the presence ofnormal levels of coagulation factors, rFVIIa might augment hemostaticmechanisms by activating factor IX on the platelet surface. Thus, rFVIIamediation of thrombin generation on the platelet surface mightcompensate for a lack of platelets in thrombocytopenia or a lack ofproper platelet function in thrombasthenia. The hypothesis that rFVIIacan play a “compensating” role for platelet function(s) is supported bythe clinical observation that rFVIIa can improve hemostasis in patientsthat are thrombocytopenic (e.g., Poon et al., 2000, Kristensen et al.,1996), thrombasthenic (e.g., Al Douri et al., 2000) or are thoseaffected by both platelet defects, such as in trauma (e.g., Dutton etal., 2003). However, high-doses of rFVIIa are frequently required forhemostasis, perhaps due to the low (Kd˜90 nM) affinity of thecoagulation factor for the platelet surface (Monroe et al., 1997). Wethus hypothesize that the direct chemical tethering of rFVIIa to theplatelet surface will improve the therapeutic effectiveness (index) ofthe recombinant protein as a hemostatic agent to stop active bleeding

c) Antiviral delivery with lyophilized platelets—

RL platelets and RL RBCs hold promise for delivering antiviraltherapeutics to macrophages that are infected with the single strand RNAviruses that cause hemorrhagic fevers and hepatitis. This is importantbecause the macrophages of the reticuloendothelial system (RES) areinvolved in the initial stage of viral infection, as well as macrophagesat sites of vascular injury in the later acute-hemorrhagic phase ofinfection. Viruses of the Arenaviridea (e.g., Lassa fever virus),Filoviridae (e.g., Ebola and Marburg viruses), Bunyaviridae (e.g., RiftValley virus) and Flaviviridae (e.g., Yellow fever virus) families causeviral-induced cellular damage to vascular tissues that result inhemorrhage. Similarly, hepatitis C virus (a Flaviviridae family member)propagation is frequently associated with bleeding-intensive hepaticsurgeries. Ribavirin, as a broad-spectrum antiviral RNA mutagen, holdspromise for the treatment of these hemorrhage-associated viruses.However, adverse toxicities have limited the clinical use of thisribonucleoside as an antiviral chemotherapeutic. We seek to increase thetherapeutic efficacy of ribavirin by using RL platelets to deliver theribonucleoside. The intrinsic hemostatic function of RL platelets willthus concentrate the ribavirin in the microenvironment of the virus forincreased chemotherapeutic efficacy; in RES and vascular wound sitemacrophages.

The present invention is described in greater detail in the followingnon-limiting Examples.

Examples 1-5 Attachment of Ribavirin to Platelets

These examples describe methods for the preparation and characterizationof reconstituted platelets having ribavirin and rFVIIa coupled thereto.

Example 1 Synthesis of Ribavirin-Polylysine Polymers

Ribavirin is chemically phosphorylated for ribavirin monophosphate (RMP)as detailed by Yoshikawa et al., Tetrahedron Lett. 50, 5065-5068(1967)). Ribavirin monophosphate (RMP) is coupled to polylysine via apH-sensitive phosphoramide linkage in accordance with the procedure ofDi Stefano and Fiume (Trends in Glycosci. and Glycotech. 50, 461-472(1997)) or a simplified procedure is based on the formation of animidazole-ribavirin adduct (see Chu et al., Nuc. Acids. Res. 11,6513-6529 (1983)). The conjugation chemistry for ribonucleosides polymersynthesis was tested with uracil rather than ribavirin. We do notanticipate that the difference in nitrogenous base structure betweenuracil and ribavirin will have a large effect on the synthesis.

Polylysine (<mw>=205 kDa for ˜1,400 residues lysine/molecule) wasreacted with FITC (fluorescein isothiocyanate) and SANPAH(N-succinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate) torespectively provide a fluorescent label and a photoactivatable moietyfor the covalent attachment of the final product to the platelets.Uracil was coupled to the lysine side-arms by first activating UMP withimidazole with EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as acatalysis. The activated UMP˜imidazole complex was then added to theFITC/SANPAH-modified lysine chain to form phosphoamide bonds. Based onpeak sizes from proton NMR analysis (α-carbon proton vs. nitrogenousbase protons, see FIG. 1, “Proton NMR Spectra of Nucleoside Copolymer”12% of the lysine residues were coupled to UMP. The final averagecomposition of the 1400 residue copolymer was 168, 20 and 4 lysineside-arms modified respectively with uracil, SANPAH and FITC, leaving˜1200 lysine residues unmodified.

In a separate series of reactions, epsilon amino groups of polylysine(Sigma-Aldrich, <MW>=200,000 kDa) are modified with FITC to provide afluorophoric label and with the heterobifunctional cross-linker SANPAH(Pierce) to provide a moiety for the photochemical attachment toplatelets in the next step (b). FITC and SANPAH are attached to thepolylysine for one FITC moiety/˜300 lysine residues and one SANPAHmoiety/˜30 lysine residues. RMP is imidazolated and then coupled to theFITC, SANPAH polylysine to form the phosphoramide bond for the ribavirincopolymer as detailed by Chu et. al. (supra). The goal is to saturatethe lysine residues with RMP residues. Proton and ¹³C NMR are performedto characterize the extent of polymer modification.

The pH sensitivity of the phosphoamide bond (see Fume et al., Pharm.Acta. Hely. 137-139 (1988)) is verified by incubating the ribavirincopolymer in buffers with pHs varying between 2.4 and 7.4 for one hr.After the incubation, pHs are adjusted to neutrality and dialyzed vs.PBS to remove free RMP. The samples ds examined with proton NMR todetermine the extent of hydrolysis. The rate of hydrolysis shouldincrease as the pH is lowered from neutrality.

We choose to test the conjugation chemistry for ribonucleosides polymersynthesis with uracil rather than ribavirin because of the temporaryunavailability of the antiviral immediately before and during themilitary operation in Iraq. We do not anticipate that the difference innitrogenous base structure between uracil and ribavirin will have alarge effect on the synthesis.

Example 2 Attachment of Ribavirin-Polylysine Polymers forRibavirin-Loaded Lyophilized Platelets

The procedure for preparing lyophilized platelets involves four steps:Removal of platelets from excess plasma proteins, mild paraformaldehydecross-linking to stabilize cellular structures, removal of plateletsfrom unreacted cross-linker and lyophilization (Read et al, U.S. Pat.No. 5,651,966). After removal of the excess paraformaldehyde, theplatelets are mixed with varying concentrations of the ribavirin polymerand exposed to visible light to activate the SANPAH moieties for theformation of covalent linkage with the platelet surface. The plateletsare then lyophilized with standard procedures. Platelets are preparedwith different amounts of ribavirin by varying the concentration ofribavirin-copolymer in the coupling step. The ability of theribonucleoside copolymer to covalently couple to RL platelets wasstudied by incubating the cells with the delivery polymer invisible-spectrum light to photoactivate the SANPAH moiety for covalentcoupling. Unreacted copolymer was removed with centrifugational washing,and then the platelets were examined with fluorescent microscopy. FIG.2, “Fluorescent Microscopy of Polymer-Modified RL Platelets”,demonstrates that he fluorescent-labeled ribonucleoside copolymerassociated with the cells. Based on the average fluorescent intensity ofthe platelets, each cell was covalently labeled with 24,800 copolymers.

Example 3 Characterization of Ribavirin-Platelets

Ribavirin-platelets prepared in accordance with example 2 above arerehydrated with normal saline and then subject to analysis to verifychemical nature of the copolymer attachment, the functionality of theplatelets and the ability of the ribavirin-platelets to release theribonucleoside.

Surface Density and Cellular Distribution of the Ribavirin-PolylysinePolymer.

Ribavirin-loaded platelets are subjected to liquid scintillationcounting, and then the amount of attached [³H]ribavirin-copolymer perplatelet calculated from the specific activity of the radiolabel. Flowcytometry may also be performed to assess the relationship betweenfluorescence intensity and the number of copolymers per platelet. Thecellular localization of the [³H]ribavirin-copolymer may be ascertainedwith confocal microscopy from the fluorescence of the FITC moiety.

The chemical integrity of the ribavirin units is verified by placing theribavirin-platelets in low pH buffer to hydrolyze the nucleoside.Samples will then be subjected to TLC analysis (as detailed in Fischeret al, Brit. J. Haem. 111, 167-175 (2000)) to determine if the[³Hhydrolysis product co-elutes with [³H]ribavirin monophosphatestandards.

The Activation Response of Ribavirin-Platelets.

Platelets loaded with increasing amounts of [³H]ribavirin-copolymer aresubjected to the same type of analysis that was utilized to characterizereconstituted lyophilized platelets prepared for transfusion purposes.Briefly, the morphology of activated and unactivated ribavirin-plateletsis examined with scanning and transmission electron microscopy.vWf-mediated adhesion may be investigated with ristocetin aggregation.The surface density of phosphatidylserine, p-selectin, activatedGPIIb/IIIa and fibrinogen may be ascertained with flow cytometry. Theseprocedures may be used to confirm the maximum degree of copolymermodification that can be obtained without unduly adversely affectinghemostatic function and hence optimize any particular preparationprotocol.

Endocytosis of Ribavirin-Platelets by Macrophages for Drug Release andAntiviral Activity.

This analysis utilizes a tissue culture-based splenic macrophagephagocytosis system that has been detailed elsewhere (Fischer et al.,Art. Cell. Blood Subs. Imm. Biotech. 29, 439-451 (2001)). The antiviralactivity of ribavirin-RL platelets may be assessed by infecting the ratsplenic macrophages with the Adames strain of Punta Toro virus, since itcan be handled under level 2 biosafety conditions, is known to infectrodent macrophages, causes a lethal hepatic necrosis in infected mice,and is sensitive to the antiviral effects of ribavirin in vivo and invitro. Alternatively these studies can optionally be adapted to mousemacrophage cultures, which are known to be highly permissive for PuntaToro virus replication.

The [³H]ribavirin-copolymer-RL platelets are incubated with themacrophages with gentle rocking at 37° C. Samples are withdrawn as afunction of time and subjected to two types of analysis to respectivelyfollow a) platelet internalization for viral inactivation and b)ribavirin metabolism.

Platelet Phagocytosis and Viral Inactivation.

These studies employ fluorescent microscopic and flow cytometricanalysis to identify macrophages (PE fluorescence fromanti-macrophage-PE) and ribavirin-RL platelet phagocytosis (from[³H]ribavirin-copolymer FITC fluorescence). The cell mixtures areincubated with anti-macrophage-PE conjugate (MCA-342, Serotec) to labelthe macrophages, then flow cytometry and confocal fluorescencemicroscopy are performed to follow the internalization and phagocyticprocesses. Flow cytometry may be relied upon to provide quantitativedata, while confocal microscopy may be performed to examine thedistribution of the ribavirin-platelets that are docked to and/orinternalized by macrophages.

A subset of macrophages treated with ribavirin-RL platelets or controlplatelets may be infected in triplicate with Punta Toro virus at amultiplicity of infection of 10 for one hour at 37° C. At this point,the cells are washed 3 times with room temperature growth medium, andmaintained in growth medium at 37 degrees C. After completing thewashes, a sample of growth medium is removed from each well for analysisof viral levels by plaque assay. Additional samples are removed atfour-hour intervals to determine levels of virus within the supernatant.Viral titers will be assessed by plaque assays on Vero cells, which arepermissive for Punta Toro virus infection and have been used previouslyto titer Punta Toro virus levels by plaque assay. Alternatively, cellsare infected with Punta Toro virus prior to treatment with ribavirin-RLor control platelets. This will assess whether ribavirin-RL plateletscan control previously established infections. As a positive control forthese studies, groups of macrophages are infected with Punta Toro virusin triplicate. These cultures are then treated with ribavirin (notloaded on platelets) at a concentration ranging from 4-10 micrograms perml, a concentration range that has previously been shown to inhibitPunta Toro virus replication in vitro. Ribavirin-RL platelets areconsidered to exhibit antiviral effect if they decrease viral titers intreated macrophage cultured to levels that are comparable to thoseobserved with conventional ribavirin treatment.

Ribavirin Metabolism.

The time course of [³H]ribavirin-monophosphate release and metabolism isexamined by lysing cell mixtures with TX-100 and then resolving freenucleosides with TLC analysis (Fischer et al., 2000, supra). Thisinformation is used to verify the release of [³H]ribavirin monophosphatefrom the copolymer as well as the conversion to[³H]ribavirin-diphosphate and triphosphate.

Example 4 Characteristics of Platelets

Preferably platelets with at least 100,000 ribavirin copolymers per cellthat function with 90% efficiency in the ristocetin aggregation analysisare obtained by the procedures described above (or modifications thereofwhich will be readily apparent to those skilled in the art). Thesecriteria are chosen for two reasons. First, a 10% reduction in theristocetin in vitro hemostasis parameter has an insignificant effect onin vivo hemostatic function of RL platelets as judged by the ability tocorrect bleeding in thrombocytopenic rabbits. Second, the delivery of100,000 ribavirin-copolymer molecules on a single platelet represents3×10⁸ ribavirin moieties (or ˜5×10⁻¹⁶ moles of ribavirin). If a singlemacrophage (with an intracellular volume of ˜10³ um³) internalizes asingle ribavirin-platelet, the intracellular concentration of ribavirinfunctions will be ˜5×10⁻¹⁹ moles/um³ or ˜5×10⁻⁴ moles/liter (500 uM).This is a concentration that is well above the range of ribavirin(10-100 uM) that exhibits antiviral activity in tissue culture (see e.g.Crotty et al., J. Mol. Med. 80, 86-95 (2002)).

The in vitro metrics that are most correlative with in vivo hemostaticefficacy for RL platelets are related to GPIb-von Willebrand factormediated adhesion. We thus utilized ristocetin-mediated aggregation forthe preliminary functional characterization of theribonucleoside-labeled RL platelets. FIG. 3, “Ristocetin Aggregation ofControl and Polymer Modified RL Platelets” demonstrates that afterrehydration, the labeled cells retained ˜70% of functionality as judgedby the initial slope and extent of aggregation curves.

Example 5 Internalization of Ribavirin in Platelets

In an alternative embodiment of the foregoing, the ribavirin is modifiedfor platelet internalization. For example, this is carried out bycoupling the short chain ribavirin polylysine copolymers to IgG orfibrinogen for alpha granule uptake. Alternatively, latex nanoparticlesare loaded with ribavirin for platelet endocytosis, and then cells willprocessed for lyophilization.

Examples 6-8 Lyophilized Platelets for Delivery of Nucleic Acids

These examples describe methods for using rehydratable, lyophilized (RL)platelets to deliver nucleic acids to sites of vascular injury for drugdevelopment, diagnostic, or therapeutic purposes. The nucleic acid mayencode any protein or peptide of interest, such as a reporter protein orpeptide for use in diagnostic or drug screening purposes.

RL platelet/AAV˜Lac Z conjugates are prepared as depicted in FIG. 4,“Preparation of Platelet/AAV˜Lac Z Reporter Conjugates” herein. Theprocedure is a variation of the procedure described in Read et al., U.S.Pat. No. 5,651,966.

Example 6 Attachment of Anti-AAV Antibodies to Platelets

Preparation of Platelets for Antibody Attachment.

Fresh porcine platelets are isolated with differential centrifugation(to obtain platelet-rich plasma) and Sepharose CL2B chromatography (tofree platelets from plasma proteins) in accordance with known techniques(Read et al., 1 Proc. Natl. Acad. Sci. USA 92, 397-401 (1995)). Thecells are then be stabilized with paraformaldehyde and washed withcentrifugation to remove unreacted cross-linker. These steps will employknown protocols that have been detailed elsewhere (Read et al., supra).

Modification and Attachment of Anti-AAV Antibody to Platelets.

Anti-AAV capsid protein VP 1 polyclonal antibody (Research DiagnosticsInc, Pleasant Hill, N.J., ALS24107) polyclonal antibody (to residues278-289 on the capsid protein) is reacted with N-succinimidyl3-[2-pyridylthio]propionate (SPDP) to form lysine-imide linkages betweenthe cross-linker and the antibody in accordance with known techniques(see Carlsson et al., Biochemistry J. 173, 723-737 (1978)). Excesscross-linker is removed with sizing chromatography, and then thethiol-activated antibody is incubated for 2 hr with the aldehydestabilized platelets prepared as above for attachment of the antibody tothe platelet surface via sulfhydryl moieties. The reaction stoichiometryis 10,000 anti-capsid antibodies per platelet. Unbound antibody isremoved with two centrifugation washes.

Attachment of AAV˜Lac Z to Platelets for Lyophilized Platelet/AAV˜Lac ZConjugates.

AAV˜Lac Z is prepared with “triple-plasmid” transfection methods inwhich 293 cells are co-transfected with three plasmids: pAAV with Lac Z,a second plasmid with Rep and Cap genes, and a third plasmid thatencodes adenovirus proteins that mediate the “rescue” step (see Monahanand Samulski, J. Virol. 61, 3096-3950 (2000)). With this strategy, the293 cells do not have to be infected with adenovirus to “rescue” therecombinant AAV particles, thus avoiding adenoviral contamination of thefinal AAV preparation. AAV˜Lac Z is isolated from the 293 cell tissueculture with ammonium sulfate precipitation and density gradientpurification in accordance with known techniques.

To attach the AAV˜Lac Z to the platelet surface, the platelets areincubated for three hours with the platelet˜anti-capsid IgG conjugatesprepared as above. Excess AAV˜Lac Z is removed with two centrifugationwashes. The platelet/AAV˜Lac Z conjugates are then frozen, lyophilizedand stored at −20° C. The freeze-dried platelet/AAV˜Lac Z conjugates areprepared so that upon rehydration, the platelet and AAV˜Lac Zconcentrations will respectively be 1×10⁸ platelets/ml and 1×10¹²AAV˜Lac Z vectors/ml.

Example 7 Characterization of Platelet/AAV˜Lac Z Conjugates

Surface Density of AAV˜Lac Z on the Platelets.

The amount of anti-capsid protein antibody and AAV˜Lac Z that isattached to the platelet surface is quantified by subjecting samples toSDS-PAG electrophoresis and Western analysis. Anti-rabbit IgG is used todetect the anti-AAV, while anti-Capsid VP1 is used to probe for AAVproteins. The object is to attach approximately 100,000 AAV˜Lac Zvectors to each platelet. If a lower stoichiometry is obtained by anyparticular procedure the concentration of the antibody can be adjustedupwards, and/or the incubation time for the platelet reaction withAAV˜Lac Z may be increased.

The Effect of Lac Z Attachment on Platelet Function.

The effect of platelet surface modification with AAV˜Lac Z on RLplatelet function may be ascertained by performing aggregation assaysand Baumgardner analysis in accordance with known techniques (Khandelwalet al., FASEB J 11, 1812 (1997); Bode et al. J. Lab. Clin. Med 133,200-211 (1999)).

The Effect of AAV˜Lac Z Attachment on Transduction Efficiency.

The effect of surface attachment on AAV˜Lac Z transduction efficiency isestimated by incubating the AAV˜Lac Z conjugates with porcine splenicmacrophages and then probing for gene transfer by staining with X-gal.Control experiments may be performed with titers of AAV˜Lac Z that areequivalent to the number of virus particles delivered with theplatelets.

An important issue in the development of AAV as a gene therapy tool isdifficulties in obtain high titer preparations (e.g., see Monahan andSamulski, supra). By immuno-absorbing AAV˜Lac Z to the surface of theplatelet, a very high local concentrations of the probe will beproduced.

The coupling of AAV to platelets may affect the function of both theviral and platelet systems. If the performance of the platelets inBaumgardner and aggregation assays is measurably affected by the probeattachment, the surface density of the AAV˜Lac Z may be reduced. Ifplatelet-bound AAV is found to transduce splenic macrophages with adramatically reduced efficiency, the surface density of the viral vectormay be increased. Alternatively, other viral tethering strategies may beutilized.

Example 8 Reporter Gene Delivery to Sites of Vascular Injury

The ability of RL platelet/AAV˜Lac Z conjugates to transduce the Lac Zgene into sites of vascular injury will be investigated. In this exampleatherosclerotic vessels in pigs on an atherogenic diet are injured, RLplatelet/AAV˜Lac Z conjugates are infused, and the animals continued onan atherogenic diet to produce additional plaque development at thesites of injury. This design models the sequence of events that occurwhen a vessel is reoccluded with atherosclerotic plaque after theendothelium is perturbed by atherosclerotic plaque rupture and/orangioplasty. A detailed description of the experiment follows. Thissystem is useful in testing drugs such as statins or the like, ordietary intervention, for activity in treating or relievingatherosclerosis in a subject.

Induction of Atherosclerosis.

Four Lpb^(1/1) genotype 30-40 kg (young adult) male pigs are used. TheLpb^(1/1) (Lpg=L(lipoprotein), b(apoB 100), p(pig)) is a polymorphgenotype that has been bred in a closed colony at the Francis Owen BloodResearch Laboratory at UNC-Chapel Hill. Inherited in an autosomalco-dominant fashion, the Lpb^(1/1) animals develop a stable degree ofhypercholesterolemia on an atherogenic diet and consistently developmoderately severe atherosclerosis that correlates closely with dietarycholesterol levels (Nichols et al., Am. J. Pathyol. 140, 403-415(1992)). Two pigs are placed on a high cholesterol diet and two remainon a standard low-cholesterol feed.

Vascular Injury and Treatment with RL Platelet/Lac Z Constructs.

After two months on the control or atherogenic diet, the animals areanesthetized and portions of the right femoral artery surgicallyisolated. Sites of vascular injury are formed by crushing the arterywith a Goldblatt clamp at two cm intervals. Five wounds are establishedover a 10 cm length of vessel. Immediately after establishing thewounds, 10⁹ AAV˜Lac Z modified platelets are rehydrated in 10 ml sterilesaline and infused into the peripheral ear vein. The surgical incisionsare repaired, and then the animals maintained on control orhigh-cholesterol diets for two additional months.

Post-Mortem Analysis of Tissues.

After the post-surgery two months on the control or high-cholesteroldiet, the animals are euthanized. The injured portion of the femoralartery, as well as similar portions of the contralateral vessel, arehistochemically examined with X-gal staining to probe for the expressionof the Lac Z gene product galactosidase. Spleen, lung, cardiac and livertissue may also be analyzed for expression of the transgene.

Example 9 Coagulation Protein Conjugation and Delivery to a VascularWound Site

The preparation of a coagulation protein-platelet conjugate can utilizea heterobifunctional cross-linker such as SANPAH(N-succinimidyl6-[4″-axido-2′-nitrophenylamino]hexanoate) with a primary amine reactiveand photoactivatable moieties for covalent attachment. Variouscoagulation proteins can be used, including recFVIIa (Novo Nordiskproduct), FVII or FVIIa and related mutants from various expressionsystems, as well as coagulation proteins that generate FVIIa, such asFXIIa or FXa. To prepare a RL platelet-redFVIIa conjugate, a stocksolution of 100 mM SANPAH is prepared in anhydrous DMSO, then diluted1/100 into 1 mg/ml recFVIIa (e.g., from Novo Nordisk) in phosphatebuffered saline (10 mM phosphate, 150 mM NaCl, pH=7.4) and allowed toincubate in the dark for 1 hr. Unreacted SANPAH is removed by dialyzingthe mixture overnight vs. PBS or gel filtration on Sepharose CL4B.

Platelets are subjected to aldehyde stabilization and then freed fromexcess paraformaldehyde as detailed in U.S. Pat. No. 5,651,966, and thenthe SANPAH-recFVIIa is conjugated to the cells. The SANPAH-recFVIIa andthe fixed platelets are then mixed for 100,000 platelets/ul and 0.1mg/ml SANPAH-protein conjugate in PBS, and then exposed to visible rangelight from a standard fluorescent source for 1 hour to achievephotocoupling of the SANPAH-recFVIIa conjugate to the platelet surface.Unreacted protein is then separated from the platelets withchromatography on Sepharose CL-2B, and then the recFVIIa-plateletconjugates are lyophilized as detailed in U.S. Pat. No. 5,651,966.

Example 10 Coupling of Recombinant Factor VIIa to Surface of LyophilizedPlatelets Through Native Binding Sites

Preparation of fluorescein Isothiocyanate (FITC) labeled recombinantfactor VIIa (rFVIIa)-4.8 mg rFVIIa (Novallordisk, infusion grade)rehydrated with 4.8 ml dist. H₂O for [rFVIIa]=20 uM. rFVIIa was dialyzedovernight vs phosphate buffered saline (PBS), then incubated for 30 minat r.t. with 40 uM FITC. The reaction mixture was dialyzed overnight vsPBS, then overnight again vs. citrated saline to obtain rFVIIa-FITC.

Surface attachment of rFVIIa-FITC to rehydrated, lyophilizedplatelets-Lyophilized platelets were hydrated with distilled water for1.89×10⁹ cells/ml. Platelets and rFVIIa-FITC were mixed and incubated atroom temperature for 30 minutes at varying concentrations ranging from 0to 10 uM rFVIIa-FITC and 0 to 1.89×10⁹ cells/ml. The platelets werecentrifugally washed once with saline, then diluted into PBS+2%paraformaldehyde for flow cytometric analysis.

rFVIIa-FITC bound to lyophilized platelets in a homogeneous manner basedon the symmetrical flow-cytometric histogram (see FIG. 5, “Flowcytometric analysis of rFVIIa-FITC bound to RL platelets).

The degree of binding of rFVIIa-FITC to lyophilized platelets was anincreasing function of total rFVIIa-FITC concentration (see FIG. 6,“rFVIIa binding to RL:platelets is concentration dependent”).

These results show that significant amounts of rFVIIa can be attached tolyophilized platelets through simple co-incubation atsuper-physiological concentrations.

The activity of rFVIIa that is surface-bound to lyophilized plateletswas analyzed by measuring the ability of these preparations to catalyzeprothrombin to thrombin conversion. RL platelet-rFVIIa was prepared byincubating rFVIIa (10 uM, 3 uM, 1 uM 0.3 or 0 uM) with RL platelets(1×10⁵/u1) in citrated saline with 10 mM CaCl₂ for one hour. RLplatelets-rFVIIa particles were centrifically washed once to removeunbound rFVIIa, then the preparations (as well as control buffer orsimilar concentrations of rFVIIa alone) were diluted 1/10 into normal orfactor IX deficient plasma that contained the fluorometric thrombinsubstrate D-phe-pro-arg-ANSNH. The time for initiation and maximal rateof thrombin substrate generation was measured. The results in FIG. 7show that RL platelet-bound rFVIIa is at least as active as free rFVIIaon a molar basis (See FIG. 7, “RL platelet-rFVIIa and free rFVIIacatalyze IIa generation in a similar manner”.

REFERENCES

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. Fixed-dried blood cells having an exogenousactive agent coupled thereto, wherein said fixed-dried blood cellscomprise fixed-dried blood platelets; wherein said exogenous activeagent is selected from the group consisting of diagnostic agents andtherapeutic agents; wherein said active agent is coupled covalently tothe interior or surface of said cells; wherein said fixed-dried bloodplatelets are aldehyde fixed-dried blood platelets; and wherein saidfixed-dried blood platelets upon reconstitution: adhere to thrombogenicsurfaces; undergo shape change upon adhering to a thrombogenic surface;and lead to the formation of a hemostatic plug upon adhering to athrombogenic surface.
 2. The fixed-dried blood cells of claim 1, whereinsaid fixed-dried blood cells comprise mammalian fixed-dried blood cells.3. The fixed-dried blood cells of claim 1, wherein said fixed-driedblood cells comprise human fixed-dried blood cells.
 4. The fixed-driedblood cells of claim 1, wherein said active agent is contained withinsaid cells.
 5. The fixed-dried blood cells of claim 1, wherein saidfixed-dried blood cells are fixed-dried blood platelets which uponreconstitution: release their granular contents upon adhering to athrombogenic surface.
 6. The fixed-dried blood cells of claim 1, whereinsaid active agent comprises an antiviral agent.
 7. The fixed-dried bloodcells of claim 1, wherein said active agent comprises a bloodcoagulation protein.
 8. The fixed-dried blood cells of claim 1, whereinsaid active agent comprises a blood anti-coagulation protein.
 9. Thefixed-dried blood cells of claim 1, wherein said active agent comprisesa nucleic acid.
 10. The fixed-dried blood cells of claim 1, wherein saidexogenous active agent is selected from the group consisting ofdetectable compounds, antiviral agents, blood coagulation proteins,blood anti-coagulation protein, and nucleic acids
 11. A pharmaceuticalcomposition comprising: a pharmaceutically acceptable carrier; andfixed-dried blood cells having an exogenous active agent coupledthereto, said fixed-dried blood cells rehydrated in saidpharmaceutically acceptable carrier, wherein said fixed-dried bloodcells comprise fixed-dried blood platelets; wherein said exogenousactive agent is selected from the group consisting of diagnostic agentsand therapeutic agents; wherein said active agent is coupled covalentlyto the interior or surface of said cells; wherein said fixed-dried bloodplatelets are aldehyde fixed-dried blood platelets, and wherein saidfixed-dried platelets upon reconstitution:adhere to thrombogenicsurfaces; undergo shape change upon adhering to a thrombogenic surface;and lead to the formation of a hemostatic plug upon adhering to athrombogenic surface.
 12. The pharmaceutical composition of claim 11,wherein said carrier is a sterile carrier.
 13. The pharmaceuticalcomposition of claim 11, wherein said carrier is a solid carrier. 14.The pharmaceutical composition of claim 11, wherein said carrier is aliquid carrier.
 15. The pharmaceutical composition of claim 11, whereinsaid carrier is an aqueous carrier.
 16. The pharmaceutical compositionof claim 11, wherein said fixed-dried blood cells comprise mammalianfixed-dried blood cells.
 17. The pharmaceutical composition of claim 11,wherein said fixed-dried blood cells comprise human fixed-dried bloodcells.
 18. The pharmaceutical composition of claim 11, wherein saidactive agent is contained within said fixed-dried blood cells.
 19. Thepharmaceutical composition of claim 11, wherein said fixed-dried bloodcells are fixed-dried platelets which upon reconstitution: release theirgranular contents upon adhering to a thrombogenic surface.
 20. Thepharmaceutical composition of claim 11, wherein said active agentcomprises an antiviral agent.
 21. The pharmaceutical composition ofclaim 11, wherein said active agent comprises a blood coagulationprotein.
 22. The pharmaceutical composition of claim 11, wherein saidactive agent comprises a blood anti-coagulation protein.
 23. Thepharmaceutical composition of claim 11, wherein said active agentcomprises a nucleic acid.
 24. The pharmaceutical composition of claim11, wherein said exogenous active agent is selected from the groupconsisting of detectable compounds, antiviral agents, blood coagulationproteins, blood anti-coagulation protein, and nucleic acids.
 25. Amethod of making fixed-dried blood cells having an exogenous activeagent coupled thereto, comprising: providing fixed blood cells, saidfixed blood cells comprising fixed blood platelets; coupling saidexogenous active agent covalently to the interior or surface of saidfixed blood cells; and drying said fixed blood cells to producefixed-dried blood cells carrying said active agent; wherein saidexogenous active agent is selected from the group consisting ofdiagnostic agents and therapeutic agents; wherein said fixed-dried bloodplatelets are aldehyde fixed-dried blood platelets, and wherein saidfixed-dried blood platelets upon reconstitution: adhere to thrombogenicsurfaces; undergo shape change upon adhering to a thrombogenic surface;and lead to the formation of a hemostatic plug upon adhering to athrombogenic surface.
 26. The method of claim 25, wherein saidfixed-dried blood cells carrying said active agent are rehydrated in apharmaceutically acceptable carrier to provide a pharmaceuticallyacceptable composition comprising rehydrated fixed-dried blood cellscarrying said active agent.
 27. The method of claim 25, wherein saidfixed blood cells comprise fixed mammalian blood cells.
 28. The methodof claim 25, wherein said fixed blood cells comprise fixed human bloodcells.
 30. The method of claim 25, wherein said associating step iscarried out by covalently or non-covalently coupling said active agentto said fixed blood cells.
 31. The method of claim 25, wherein saidassociating step is carried out by introducing said active agent intosaid fixed blood cells.
 32. The method of claim 25, wherein saidrehydrated fixed-dried blood cells comprise rehydrated fixed-dried bloodplatelets which: release their granular contents upon adhering to athrombogenic surface.
 33. The method according to claim 25, wherein saidexogenous active agent is selected from the group consisting ofdetectable compounds, antiviral agents, blood coagulation proteins,blood anti-coagulation protein, and nucleic acids.